Respiratory viral infections remain a major health concern causing morbidity and mortality, especially in elderly and immunocompromised individuals. Current vaccines against influenza rely on inducing neutralizing antibodies to coat proteins;however, these vaccines are ineffective against serologically distinct viral strains and strains that have pandemic potential. Therefore, it is of extreme importance to develop influenza vaccines that target T cell immunity, however, the mechanisms that direct T cell trafficking to the lung and the T cell effector mechanisms that correlate with protection have not been fully defined. In addition, it is not known which adjuvants or vaccine preparations are best suited to prime effective T cell responses that persist to memory. To address these gaps in our knowledge, we will use a mouse model of influenza infection and CD4 T cell receptor (TCR) transgenic (tg) mice specific for influenza H1N1 to track the development of CD4 T cell effectors that traffic to the lung, persist to memory and contribute to protection following vaccination. Our previous studies and preliminary data demonstrate that CD4 T cells acquire perforin mediated cytotoxicity following influenza infection and protect against lethal challenge in an antibody independent manner. The objective of this proposal is to determine which vaccine strategies elicit cytolytic CD4 T cells that migrate to the lung and afford protection to lethal influenza infection. We will focus on toll like receptor (TLR) ligands as adjuvants because of their ability to mobilize dendritic cells (DC), potent activators of T cell responses. In Aim 1, we will determine the role of TLR ligands in promoting class II restricted cytolytic activity in vitro and in vivo. The first approach will be to isolate DC populations from the lung, activate with TLR ligands, co-culture with TCR Tg CD4 cells and determine if cytolytic CD4 cells are generated in vitro. The second approach will be to confirm the adjuvant effect of TLR agonists in vivo by transferring naive TCR Tg CD4 cells into mice, vaccinating via the respiratory tract and analyzing the CD4 response in the lung. In Aim 2 we will determine whether vaccine induced cytolytic CD4 cells differentiate to the memory stage and contribute to protection against lethal influenza infection. We will use the TLR ligands that elicit cytolytic CD4 cells as a correlate of protection and test whether those adjuvants induce a memory population and provide protection against a lethal influenza challenge. Finally, using perforin deficient CD4 T cells and B cell deficient mice, we will test whether perforin dependent cytolytic activity is required for antibody independent protection mediated by CD4 T cells. PUBLIC HEALTH RELEVANCE: Respiratory infections such as influenza pose a major public health threat. The proposed studies will further our understanding of how CD4 T cell immunity develops and persists in the lung in response to vaccination with adjuvant preparations that activate the innate immune response. This information will be important for developing novel vaccine strategies that target cell mediated immunity and provide more universal protection against emerging, serologically distinct influenza strains such as H5N1 and H1N1.